Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• This invention pertains to liposomal formulations of mitoxantrone and methods for their manufacture and use.
• Mitoxantrone is a therapeutic agent which is useful for the treatment of cancer and multiple schlerosis.
• the U.S. Food and Drug Administration (FDA) first approved mitoxantrone hydrochloride for sale in the United States in 1987 as an injectable formulation under the tradename Novantrone®.
• Novantrone® is provided as a sterile, nonpyrogenic, dark blue aqueous solution containing an amount of the hydrochloride salt form equivalent to 2 mg/ml mitoxantrone free base, with sodium chloride (0.80% w/v), sodium acetate (0.005% w/v), and acetic acid (0.046% w/v) as inactive ingredients.
• Novantrone® in combination with corticosteroids is approved for use as initial chemotherapy for the treatment of patients with pain related to advanced hormone-refractory prostate cancer.
• the recommended dosage of Novantrone is 12 to 14 mg/m 2 given as a short intravenous infusion every 21 days.
• Novantrone is also approved for use, in combination with other approved drug(s), in the initial therapy of acute nonlymphocytic leukemia (ANLL), including myelogenous, promyelocytic, monocytic, and erythroid acute leukemias.
• ANLL acute nonlymphocytic leukemia
• the recommended dosage is 12 mg/m 2 of Novantrone daily on days 1-3 given as an intravenous infusion along with 100 mg/m 2 of cytarabine for 7 days given as a continuous 24-hour infusion on days 1-7.
• Novantrone® is also approved for use in reducing neurologic disability and/or the frequency of clinical relapses in patients with secondary (chronic) progressive, progressive relapsing, or worsening relapsing-remitting multiple sclerosis.
• Mitoxantrone hydrochloride is thought to be a DNA-reactive agent that is cytotoxic to both proliferating and non-proliferating human cells in culture.
• mitoxantrone limits the dosage of drug that can be administered to patients. Moreover, the development of multidrug resistance in cells exposed to mitoxantrone can limit its effectiveness. Consequently, formulations of mitoxantrone are needed that sufficiently solubilize mitoxantrone while maximizing its efficacy for example, by minimizing toxicity and the development of multidrug resistance in treated cells.
• the present invention is for novel mitoxantrone compositions, their preparation methods, and their use in treating diseases such as cancer, particularly in mammals, especially humans.
• the method involves administering a therapeutically effective amount of the pharmaceutical composition of mitoxantrone in a pharmaceutically acceptable excipient to a mammal.
• the compositions of the present invention include liposomal formulations of mitoxantrone in which the liposome can contain any of a variety of neutral or charged liposome-forming materials and a compound such as cardiolipin that is thought to bind mitoxantrone.
• the liposome-forming material can be an amphiphilic molecule such as a phospholipid like phosphatidyl choline, dipalmitoyl phosphatidyl choline, phosphatidyl serine, cholesterol, and the like that form liposomes in polar solvents.
• the cardiolipin in the liposomes can be derived from natural sources or synthetic. Depending on the composition of the liposomes, the liposomes can carry net negative or positive charges or can be neutral. Preferred liposomes also contain tocopherol. Although a wide range of concentrations of mitoxantrone can be used in this formulation, the most useful concentrations range from 0.5 to 2 mg/ml.
• the molar ratio of the mitoxantrone to lipid component can also vary widely but the most useful range is from about 1:10 to about 1:20.
• the liposomes can be passed through filters of various sizes to control their size, as desired.
• the liposomal compositions can be used advantageously in conjunction with secondary therapeutic agents other than mitoxantrone, including antineoplastic, antifungal, antibiotic among other active agents.
• the liposomes of the present invention can be multilamellar vesicles, unilamellar vesicles, or their mixtures, as desired. Methods are provided in which a therapeutically effective amount of the present liposomes in a pharmaceutically acceptable excipient are administered to a mammal, such as a human.
• a quantity of mitoxantrone in a pharmaceutically acceptable excipient (such as Novantrone®, is added to a vessel containing a quantity of preformed lyophilized liposomes that contain a mitoxantrone-binding component, and the mitoxantrone is allowed to bind to the liposomes to provide the pharmaceutical dosage form.
• a pharmaceutically acceptable excipient such as Novantrone®
• the present invention provides a composition and methods for its manufacture and delivery to a mammalian host.
• the composition and method are characterized by avoidance of solubility problems of mitoxantrone, high mitoxantrone and liposome stability, ability to administer mitoxantrone as a bolus or short infusion in a high concentration, reduced mitoxantrone toxicity, particularly reducing mitoxantrone accumulation in cardiac muscle, increased therapeutic efficacy of mitoxantrone, and modulation of multi-drug resistance in cancer cells.
• the use of cardiolipin in the formulation improves mitoxantrone entrapment to a surprising extent.
• the inventive composition is a liposomal formulation of mitoxantrone which contains cardiolipin.
• the liposomal formulation can be prepared by known techniques. For example, in one preferred technique mitoxantrone is dissolved in a hydrophobic solvent with cardiolipin and the cardiolipin allowed to form complexes with mitoxantrone.
• the cardiolipin/mitoxantrone-containing mixture can be evaporated to form a film in order to facilitate complex formation. Thereafter, solutions containing any desired additional lipophilic ingredients can be added to the film and the mitoxantrone/cardiolipin complexes dissolved or thoroughly dispersed in the solution. The solution can then be evaporated to form a second lipid film.
• a polar solvent such as an aqueous solvent, can then be added to the lipid film and the resulting mixture vigorously homogenized to produce the present inventive liposomes.
• all of the lipophilic ingredients can be dissolved in a suitable solvent that can then be evaporated to form a lipophilic film.
• a polar solvent such as an aqueous solvent can then be added to the lipid film and the resulting mixture vigorously homogenized to produce the present inventive liposomes.
• the dosage form can be conveniently packaged in a single vial to which a suitable aqueous solution can be added to form the liposomes.
• a two vial system can be prepared in which the lipophilic ingredients or preformed liposomes are contained in one vial and aqueous ingredients containing mitoxantrone are provided in a second vial.
• the aqueous mitoxantrone-containing ingredients can be transferred to the vial containing the lipid film or preformed liposomes and the liposomal formulation of mitoxantrone formed by vigorous mixing, vortexing and/or sonicating.
• the liposomes are filtered through suitable filters to control their size.
• suitable filters include those that can be used to obtain the desired size range of liposomes from a filtrate.
• the liposomes can be formed and thereafter filtered through a 5 micron filter to obtain liposomes having a diameter of about 5 microns or less.
• 1 ⁇ m, 500 nm, 200 nm, 100 nm, or other filters can be used to obtain liposomes having corresponding sozes.
• mitoxantrone is dissolved in a suitable solvent.
• suitable solvents are those in which mitoxantrone is soluble and which can be evaporated without leaving pharmaceutically unacceptable amounts of pharmaceutically unacceptable residue.
• non-polar, slightly polar, or polar solvents can be used, such as ethanol, methanol, chloroform, acetone, or saline, and the like.
• cardiolipin can be purified from natural sources or can be chemically synthesized, such as tetramyristylcardiolipin. Cardiolipin can be dissolved in a suitable solvent, which include solvents in which cardiolipin is soluble and which can be evaporated without leaving pharmaceutically unacceptable amounts of pharmaceutically unacceptable residues.
• the cardiolipin solution can be mixed with the mitoxantrone.
• cardiolipin can be dissolved directly with mitoxantrone. It has been found that by incorporating cardiolipin in liposomes, the liposomes capacity for mitoxantrone is increased to a surprising extent.
• suitable cardiolipin derivatives can also be used in the present liposome formulation so long as the resulting liposome formulation is sufficiently stable for therapeutic use and has a suitable capacity for mitoxantrone.
• Suitable liposome-forming materials include synthetic, semi-synthetic (modified natural) or naturally occurring compounds having a hydrophilic portion and a hydrophobic portion. Such compounds are amphiphilic molecules and can have net positive, negative, or neutral charges.
• the hydrophobic portion of liposome forming compounds can include one or more nonpolar, aliphatic chains, for example, palmitoyl groups.
• suitable liposome-forming compounds include phospholipids, sterols, fatty acids, and the like.
• Preferred liposome-forming compounds include cardiolipin, phosphatidyl choline, cholesterol, dipalmitoyl phosphatidyl choline, phosphatidyl serine, and ⁇ -tocopherol.
• the liposome-forming material can be dissolved in a suitable solvent, which can be a low polarity solvent such as chloroform, or a non-polar solvent, such as n-hexane, in which it is soluble.
• suitable solvents only include solvents in which the liposome-forming material is soluble and which can be evaporated without leaving pharmaceutically unacceptable amounts of pharmaceutically unacceptable residues.
• Other components can be mixed in with this solution, including mitoxantrone, to form a solution in which all ingredients are soluble and the solvent can then be evaporated to produce a homogeneous lipid film.
• Solvent evaporation can be by any suitable means that preserves the stability of mitoxantrone and other lipophilic ingredients.
• Suitable liposomes can be neutral, negatively, or positively charged, the charge being a function of the charge of the liposome components and pH of the liposome solution.
• positively charged liposomes can be formed from a mixture of phosphatidyl choline, cholesterol, and stearyl amine.
• Negatively charged liposomes can be formed, for example, from phosphatidyl choline, cholesterol, and phosphatidyl serine.
• the liposomal mitoxantrone formulation contains tetramyristoyl cardiolipin, cholesterol, and egg phosphatidylcholine.
• the preferred liposomal mitoxantrone formulation contains suitable relative molar amounts of mitoxantrone to lipid. Suitable relative molar amounts of mitoxantrone to lipid range of about 1:1-50, more preferably, about 1:2-40, more preferably about 1:5-30, still more preferably about 1:10-20, and most preferably about 1:15.
• the liposomal formulation also contains suitable relative molar amounts of cardiolipin, phosphatidylcholine, and cholesterol.
• suitable relative molar amounts include about 0.1-25:1-99:0.1-50 of cardiolipin:phosphatidylcholine:cholesterol. More preferably, relative molar amounts range from 0.2-10:2-50:1-25, still more preferably 0.5-5:4-25:2-15, and still more preferably the amounts range from 0.75-2:5-15:4-10, the most preferred ratio being 1:10:6.8.
• Preferred liposomal formulations also contain suitable amounts of antioxidants such as ⁇ -tocopherol or other suitable antioxidants. Suitable amounts range from about 0.001 or more to about 5 wt. % or less.
• Liposomes can be formed by adding a polar solution preferably an aqueous solution, such as a saline solution, to the lipid film and dispersing the film with vigorous mixing.
• a polar solution preferably an aqueous solution, such as a saline solution
• the polar solution contains mitoxantrone.
• the solution can be pure water or it can contain salts, buffers, or other soluble active agents.
• Any method of mixing can be used provided that the chosen method induces sufficient shearing forces between the lipid film and polar solvent to strongly homogenize the mixture and form liposomes.
• mixing can be by vortexing, magnetic stirring, and/or sonicating.
• Multilamellar liposomes can be formed simply by vortexing the solution. Where unilamellar liposomes are desired a sonication and/or filtration step can be included in the process.
• a vial of lyophilized liposomes is prepared and Novantrone® is added to form the liposomal formulation of the mitoxantrone.
• the lyophilized liposomes are manufactured by dissolving the lipid ingredients and D- ⁇ -tocopheryl acid in warm butyl alcohol as described in more detail in Example 7. Warm water with trehalose dihydrate is mixed into this solution until the solution is clear. The solution is sterile filtered through a 0.22 ⁇ m filter into sterile vials and lyophilized.
• the lyophilized product is an off-white cake or powder having a moisture content of about 12% or less and that can easily be reconstituted into a uniform solution of liposomes having a pH of from about 3 to about 6.
• the final dosage form is prepared by adding 7.5 ml of a mitoxantrone solution (15 mg) such as from a Novantrone® vial and 7.5 ml of normal saline (0.9% NaCl) to a vial of the lyophilized lipids.
• the liposome mixture hydrates at room temperature for 30 minutes and is vortexed vigorously for 2 minutes at room temperature.
• the mixture is allowed to hydrate while being sonicated at maximum intensity for 10 minutes in a bath-type sonicator.
• This final dosage form may be dispensed in either a syringe or standard infusion set over 45 minutes for use within 8 hours after reconstitution. Using this method about 70 wt.
• % or more of the added mitoxantrone can be entrapped in the liposomal formulation. More preferably, about 80 wt. % or more of the mitoxantrone is entrapped. More preferable, about 85 wt. % or more of the mitoxantrone is entrapped in liposomes. Still more preferably, about 90 wt. % or more or even about 95 wt. % or more of mitoxantrone is entrapped in the liposomes.
• the efficiency of mitoxantrone entrapment can be determined by dialysis of an aliquot of the liposomal preparation overnight in an aqueous solution and thereafter dissolving the liposomes in methanol and analyzing the sample by standard methods using high pressure reverse phase liquid chromatography (HPLC).
• HPLC high pressure reverse phase liquid chromatography
• liposomes can be collected after centrifugation at 50,000 ⁇ g for 1 hour prior to dissolving them in methanol for HPLC analysis.
• the encapsulation efficiency of mitoxantrone in liposomes will be more than 80% of the initial input dose.
• any suitable method of forming liposomes can be used so long as it results in liposomal mitoxantrone.
• solvent evaporation methods that do not involve formation of a dry lipid film can be used.
• liposomes can be prepared by forming an emulsion in an aqueous and organic phase and evaporating the organic solvent.
• the present invention is intended to encompass liposomal formulations of mitoxantrone however made.
• the invention includes pharmaceutical preparations which in addition to non-toxic, inert pharmaceutically suitable excipients contain the liposomal mitoxantrone formulation and processes for production of these preparations.
• pharmaceutically suitable excipients there are to be understood solid, semi-solid or liquid diluents, fillers and formulation auxiliaries of all kinds.
• the invention also includes pharmaceutical preparations in dosage units. This means that the preparations are in the form of individual parts, for example vials, syringes, capsules, pills, suppositories, or ampoules, of which the content of liposomal entrapped mitoxantrone corresponds to a fraction or a multiple of an individual dose.
• the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1 ⁇ 2, 1 ⁇ 3 or 1 ⁇ 4 of an individual dose.
• An individual dose preferably contains the amount of mitoxantrone which is given in one administration and which usually corresponds to a whole, a half or a third or a quarter of a daily dose.
• Tablets, dragees, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays can be suitable pharmaceutical preparations.
• Suppositories can contain, in addition to the liposomal mitoxantrone, suitable water-soluble or water-insoluble excipients. Suitable excipients are those in which the inventive liposomal mitoxantrone is sufficiently stable to allow for therapeutic use, for example polyethylene glycols, certain fats, and esters or mixtures of these substances.
• Ointments, pastes, creams and gels can also contain suitable excipients in which the liposomal mitoxantrone is stable.
• the mitoxantrone formulation should preferably be present in the abovementioned pharmaceutical preparations in a concentration of about 0.1 to 50, preferably of about 0.5 to 25, wt. % of the total dry formulation.
• compositions are manufactured in the usual manner according to methods as are known, for example, by mixing the liposomal mitoxantrone with the excipient or excipients.
• the active compound and pharmaceutical preparations containing the active compound are used in human and veterinary medicine for the prevention, amelioration and/or cure of diseases, in particular those diseases caused by cellular proliferation, such as cancer, in any mammal, such as a cow, horse, pig, dog or cat.
• diseases in particular those diseases caused by cellular proliferation, such as cancer, in any mammal, such as a cow, horse, pig, dog or cat.
• dog lymphoma can be treated effectively with the present mitoxantrone formulation.
• the present formulation is particularly preferred for use in the treatment of human patients, particularly for cancer and other diseases caused by cellular proliferation.
• the inventive compositions have particular use in treating human multiple schlerosis, lymphoma, and prostate, liver, ovarian, breast, lung and colon cancers.
• the active compound or its pharmaceutical preparations can be administered locally, orally, parenterally, intraperitoneally and/or rectally, preferably parenterally, however intravenous administration is preferred.
• mitoxantrone In a human of about 70 kg body weight, for example, from about 0.5-100 mg/m 2 mitoxantrone is administered. Preferably, from about 5.0 or more to 50 mg/m 2 of mitoxantrone or more preferably from about 10 or more to about 45 mg/m 2 is administered. Still more preferably about 20 or more to about 40 mg/m 2 and still more preferably about 25 or more to about 40 mg/m 2 of mitoxantrone can be administered.
• Suitable amounts are therapeutically effective amounts that do not have excessive toxicity, as determined in empirical and case-by-case studies.
• One advantage of the present composition is that it provides a method of modulating multidrug resistance in cancer cells that are subjected to mitoxantrone treatment.
• the present liposomal formulations reduce the tendency of cancer cells subjected to chemotherapy with mitoxantrone to develop resistance thereto, and reduces the tendency of treated cells of developing resistance to other therapeutic agents, such as camptothecin, taxol, or doxorubicin, for example.
• other agents can be advantageously employed with the present treatment either in the form of a combination active with mitoxantrone or by separate administration.
• the examples demonstrate that mitoxantrone administration produces pharmacological efficacy against mammalian tumors that is not diminished by inclusion in a liposomal formulation. Further, animals could tolerate higher doses of mitoxantrone when it is administered as a liposomal formulation and they have better outcomes as measured by median survival times or reduced tumor volumes than animals given conventional mitoxantrone. Higher plasma concentrations in mice and dogs and a longer elimination half-life of compound in mice is demonstrated. Peak plasma concentrations were approximately 50-fold higher in the mouse and 9-fold higher in the dog at comparable dosages.
• This example shows one formulation of liposomal mitoxantrone.
• Mitoxantrone (3 ⁇ moles) is dissolved with cardiolipin in (3 ⁇ moles) in chloroform.
• Phosphatidyl choline 14 ⁇ moles
• 10 ⁇ moles cholesterol in chloroform is added to the mitoxantrone mixture with stirring.
• the solvents are evaporated under vacuum at about 30° C. or below to form a thin dry film of lipid and drug.
• Liposomes are formed by adding 2.5 ml of saline solution and aggressively mixing the components, as by vortexing. The flasks can then be vortexed to provide multilamellar liposomes or sonicated to provide small unilamellar liposomes.
• This example demonstrates the preparation of another formulation of liposomal mitoxantrone.
• a solution of about 6 ⁇ M mitoxantrone, 6 ⁇ M cardiolipin, 28 ⁇ M phosphatidyl choline and 20 ⁇ M cholesterol is prepared in a suitable solvent which is then evaporated.
• the dried lipid/drug film is dispersed in a 7% aqueous trehalose-saline solution.
• the mixture is vortexed and sonicated.
• the liposomes can then be dialyzed, as desired.
• Mitoxantrone encapsulation is 80% or more as assayed by HPLC.
• This example demonstrates the preparation of another formulation of liposomal mitoxantrone.
• Mitoxantrone can be entrapped in liposomes by using 3 ⁇ M of the drug, 15 ⁇ M of dipalmitoyl phosphatidyl choline, 1 ⁇ M cardiolipin, and 9 ⁇ M cholesterol in a volume of 2.5 ml.
• the drug and lipid mixture can be evaporated under vacuum and resuspended in an equal volume of saline solution.
• Liposomes are prepared as described in Example 1.
• the mitoxantrone encapsulation efficiency is higher than 80% in this system.
• This example demonstrates the preparation of another formulation of liposomal mitoxantrone.
• 2 ⁇ M mitoxantrone 2 ⁇ M of phosphatidyl serine, 11 ⁇ M phosphatidyl choline, 2 ⁇ M cardiolipin, and 7 ⁇ M cholesterol are dissolved in a solution.
• Lipiosomes are prepared as in Example 1. Greater than 80% mitoxantrone encapsulation efficiency can be expected.
• This example demonstrates another formulation of liposomal mitoxantrone.
• Mitoxantrone (3 ⁇ moles) can be dissolved in chloroform containing 3 ⁇ moles cardiolipin and the mixture allowed to form complexes. To facilitate complex formation the chloroform solvent is removed by evaporation.
• Phosphatidyl choline 14 ⁇ moles
• 10 ⁇ moles cholesterol in chloroform can be added to the dry film.
• the mixture is stirred gently and the solvents evaporated under vacuum at below 30° C. to form a thin dry film of lipid and drug.
• Liposomes are then formed by adding 2.5 ml of saline solution and aggressively mixing the components by vortexing. The flasks can then be vortexed to provide multilamellar liposomes and optionally sonicated in a sonicator to provide small unilamellar liposomes.
• This example demonstrates another formulation of liposomal mitoxantrone.
• this method involves the steps of obtaining a mitoxantrone solution, adding the mitoxantrone solution to preformed liposomes and allowing the mixture to equilibrate such that liposomal mitoxantrone forms.
• Each vial of Novantrone® contains mitoxantrone hydrochloride equivalent to 2 mg/ml mitoxantrone free base, sodium chloride (0.8% w/v), sodium acetate (0.005% w/v) and acetic acid (0.046% w/v).
• the Novantrone® solution has a pH of 3.0 to 4.5 and contains 0.14 mEq of sodium per ml.
• Preformed liposomes are prepared by adding about 2 g of D- ⁇ -tocopherol acid succinate to about 10 kg of t-butyl alcohol which is warmed to about 35-40° C. The solution is mixed for about 5 minutes until the tocopherol is dissolved. About 60 g of tetramyristoyl cardiolipin is added to the solution and the solution is mixed for about 5 minutes. About 100 g of cholesterol is added to the solution and the solution is mixed for about 5 more minutes then about 300 g of egg phosphatidyl choline is added and mixed for another 5 min. A second aqueous solution containing 2,000 g of water at about 35° C.- 40° C.
• trehalose dihydrate is mixed into the lipid solution until the mixture is clear.
• the mixture is sterile filtered through a 0.22 micron pore size Durapore® Millipak 200 filter and about 11 g is filled into sterile vials and lyophilized.
• Liposomes prepared in this manner are in the form of an off-white cake or powder and are easily reconstituted.
• the moisture content of the lyophilized liposomes is about 12% or less.
• the lyophilized product is stored at 4° C. prior to use.
• 7.5 ml mitoxantrone solution (15 mg) from a Novantrone® vial is added to a vial of lyophilized lipids along with 7.5 ml of normal saline (0.9% NaCl).
• the vial is swirled gently, allowed to hydrate at room temperature for 30 minutes, vortexed vigorously for 2 min, and sonicated for 10 min in a bath-type sonicator at maximum intensity. Doses can then be withdrawn from the vial for use.
• the product may be dispensed in either a syringe or standard infusion set over 45 min. Desirably, the liposomal mitoxantrone is maintained at room temperature until use, and is used within 8 h of reconstitution.
• This example demonstrates another formulation of liposomal mitoxantrone.
• a lyophilized lipid composition containing cardiolipin:phosphatidylcholine:cholesterol in a 1:10:6.8 molar ratio was prepared. Twenty-nine trials were conducted varying the mitoxantrone to lipid molar ratios, hydration and sonication times. Formulations were dialyzed against normal saline overnight and the amount of mitoxantrone retained in each formulation was determined.
• a 1 mg/ml mitoxantrone formulation was prepared with a 1:15 mitoxantrone to lipid molar ratio, a hydration time of 2 h, and a sonication time of 10 min.
• mitoxantrone in the liposomal formulation described above has a lower toxicity as compared to identical concentrations of nonliposomal (conventional) mitoxantrone and that at least 15 mg/kg of mitoxantrone administered in a liposomal formulation is not toxic to mice.
• Eighty male CD2F1 mice weighing 20-22 g were acclimated for 1 week and randomly separated into 8 groups of ten animals each with 5 animals per cage. On day 0 all groups of animals were injected i.v. in the tail vein with the drug or vehicle control. The volumes administered were varied based on individual animal weights. Mouse weights were recorded for each mouse on alternate days following injection and observation for clinical illness were recorded at least daily.
• blood was analyzed for bilirubin, blood urine nitrogen (BUN), creatinine, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), hemoglobin, hematocrit, white blood cell count, red blood cell count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, neutrophils, band neutrophils, lymphocytes, monocytes, eosinophils, basophils. Clinically significant elevations in ALT were noted in most of the group 1 mice and one of the group 7 mice at day 10. Similar AST elevations were also observed. Two group 1 mice also exhibited modest elevations in BUN but not creatinine, suggesting a prerenal effect, possibly caused by dehydration or hemoconcentration. No other drug related effects were observed in these studies.
• mitoxantrone in the liposomal formulation described in Example 7 has a lower toxicity as compared to identical concentrations of conventional mitoxantrone HCl and that up to 35 mg/kg of mitoxantrone can be administered to mice in a liposomal formulation without apparent toxicity.
• Twenty male CD2F1 mice weighing 20-22 g were acclimated for 1 week and randomly separated into 4 groups of five animals each with 5 animals per cage. On day 0 all groups of animals were injected i.v. in the tail vein with the drug or vehicle control. The volumes administered were varied based on individual animal weights. Mouse weights were recorded for each mouse on alternate days following injection and observation for clinical illness were recorded at least daily.
• mitoxantrone in the liposomal formulation described in Example 7 has a lower toxicity as compared to identical concentrations of conventional mitoxantrone HCl and that at least 35 mg/kg of mitoxantrone administered in a liposomal formulation is not toxic to mice.
• Seventy male CD2F1 mice weighing 20-22 g were acclimated for 1 week and randomly separated into 7 groups of ten animals each with 5 animals per cage. On day 0 all groups of animals were injected i.v. in the tail vein with the drug or vehicle control. The volumes administered were varied based on individual animal weights. Mouse weights were recorded for each mouse on alternate days following injection and observation for clinical illness were recorded at least daily.
• the mouse from group 8 also had increased alkaline phosphatase activity and the mice from groups 6 and 7 had reduced creatinine and alkaline phosphatase.
• Moribund-sacrificed mice from groups 2, 3, and 6 exhibited marked, clinically significant, compound related leukopenia with decreased neutrophils and lymphocyte counts, and a modest decrease in platelet count.
• Mice from groups 1, 4, 6, and 7 were analyzed at day 64 and exhibited moderate elevations in alkaline phosphatase and AST but where otherwise normal.
• mitoxantrone in the liposomal formulation described in Example 7 has a lower toxicity as compared to identical concentrations of conventional mitoxantrone HCl and that at least 35 mg/kg of mitoxantrone administered in a liposomal formulation is not toxic to mice.
• Thirty male CD2F1 mice weighing 20-22 g were acclimated for 1 week and randomly separated into 6 groups of five animals each with 5 animals per cage. On day 0 all groups of animals were injected i.v. in the tail vein with the drug or vehicle control and once daily thereafter for a period of 5 days. The volumes administered were varied based on individual animal weights.
• Example 8 In the single-dose experiment in Example 8, a 15 mg/kg dose of conventional mitoxantrone but not liposomal mitoxantrone induced significant increases in ALT signifying acute liver injury, but a higher dose in Example 10 did not. Taking the multiple dose data into account, it is clear that conventional mitoxantrone has the potential to cause significant liver injury. Data from the terminal sacrifices suggest that significant recovery takes place, with little evidence of either toxicity or cytotoxicity.
• mice from the higher dose groups exhibited cytotoxic effects on white blood cells and platelets, with clear decreases in neutrophils and lymphocytes and modest decreases in platelets. In the lower dose groups the effects were much less marked.
• the data show that conventional mitoxantrone at 5 mg/kg/day and liposomal mitoxantrone at 10 mg/kg/day induced roughly equivalent acute liver injury, as evidenced by increased ALT, AST and bilirubin by day 8.
• the following example demonstrates that the liposomal mitoxantrone formulation described in Example 7 reaches higher plasma concentrations, has a longer half-life, and a slower clearance rate in mammalian blood than does mitoxantrone administered in a conventional formulation.
• Pharmacokinetic evaluation was performed in male CD2F1 mice, after single dose i.v. administration of conventional and liposomal mitoxantrone formulations at 5 mg/kg. Groups of four mice were sacrificed at 5 min., 15 min., 30 min., 1 h, 2 h, 4 h, 8 h, 24 h and 48 h after dosing and their blood and organs were collected and analyzed for mitoxantrone content.
• Plasma and tissue samples were analyzed for mitoxantrone by reverse phase HPLC.
• Plasma samples (0.25 ml) were mixed with 0.5 ml of solution of 0.01 mg/ml hexanesulfonic acid, 0.5 mg/ml ascorbic acid, and 0.25 ⁇ g ametantrone as internal standard. After vortexing for 30 sec., 0.5 ml of 0.1 M borate buffer (pH 9.5) and 150 ⁇ l of 1 M sodium hydroxide was added and the solution vortexed again for 30 sec. The samples were extracted with 10 ml of dichloromethane on a horizontal shaker for 1 h and centrifuged for 15 min. at 3,000 rpm.
• Plasma Pharmacokinetic parameters were assessed by standard methods.
• the elimination rate constant (K) was calculated from the linear regression analysis of plasma concentration-time curve.
• the area under the curve (AUC 0 ⁇ ) was calculated using the linear trapezoidal method with extrapolation of the terminal phase to infinity (C last /K), where C last is the last measured concentration.
• liposomal mitoxantrone produced a significantly higher peak plasma concentration (50-fold) as compared to conventional mitoxantrone.
• the decrease in plasma concentration followed first-order kinetics with elimination half-life of 6.6 min. and 1 h for conventional and liposomal formulations, respectively.
• the AUC values and terminal elimination half-lives were C max , AUC and t 1/2 values after conventional mitoxantrone were 0.41 ⁇ g/ml, 0.14 ⁇ g•hr/ml and 0.11 hr, respectively, while these values were approximately 21 ⁇ g/ml, 28 ⁇ g•hr/ml, and 1 hr, for these same parameters after liposomal mitoxantrone administration. These increases could be explained by the decrease in both the clearance and the volume of distribution of the compound.
• the calculated total mitoxantrone clearance was substantially reduced with liposomal mitoxantrone (3 ml/min/kg) as compared to conventional mitoxantrone (600 ml/min/kg).
• the calculated volume of distribution was also markedly reduced for liposomal mitoxantrone (0.3 l/kg) versus conventional mitoxantrone (5.5 l/kg).
• This example demonstrates the efficacy of liposomal mitoxantrone, as prepared in Example 7, against human leukemia cells and demonstrates the increased efficacy of the liposomal formulation as compared to a conventional mitoxantrone formulation.
• Murine leukemia cells, L1210 leukemia cells were grown in the peritoneum of CD2F1 mice by three serial propagations (i.p.). Ascites developed within eight days of the last inoculation were used in the following experiments. Cytostatic activities of liposomal and conventional formulations of mitoxantrone against L1210 ascitic leukemia was determined. Animal group weights were determined three times a week and clinically morbid animals were humanely sacrificed. The surviving mice were observed daily for 60 days. Group survival times post i.v. treatment with single or multiple doses of the drug was indicative of the relative anti-tumor potencies of liposomal and conventional mitoxantrone.
• mice Female CD2F1 mice were divided into eight groups of 10 animals and inoculated i.v. with 10,000 L1210 cells. Drug was administered twenty-four hours later. Conventional mitoxantrone was administered at doses of 5 and 10 mg/kg. Liposomal mitoxantrone was administered i.v. at 5, 10, 20 or 35 mg/kg doses as a single injection and the median survival time for each group was determined. Surviving animals were sacrificed on day 60 of the experiment. Blank liposomes equivalent to the 35, mg/kg dose and normal saline was also administered as controls.
• the median survival time for untreated animals was 7 days. Animals treated with 5 mg/kg conventional mitoxantrone and liposomal mitoxantrone had median survivals of 12 and 13 days, respectively. The median survival time for animals given 10 mg/kg conventional mitoxantrone was 20 days, with 2/10 animals alive at day 60. The median survival time for animals treated with 10 mg/kg liposomal mitoxantrone was 27 days with 4/10 mice surviving to day 60. All animals treated with liposomal mitoxantrone at 20 mg/kg survived to day 60. At the highest dose of liposomal mitoxantrone tested, 35 mg/kg, 9/10 animals survived to Day 60, with one animal found dead on day 18, probably due to compound toxicity.
• liposomal mitoxantrone can be administered at higher doses than conventional mitoxantrone with an improved clinical outcome.
• liposomal mitoxantrone improved the median survival of animals as compared to conventional mitoxantrone at comparable dosages and decreased compound-related mortality at both the same and higher dosages.
• This example demonstrates the efficacy of liposomal mitoxantrone, as prepared in Example 7, when administered in multiple doses.
• Forty female CD2F1 mice were separated into 4 groups of ten animals and inoculated with L1210 cells as described in Example 14. The mice were treated with conventional mitoxantrone at 2.5 mg/kg or liposomal mitoxantrone at 2.5 or 5 mg/kg every 24 hours for 4 days starting 24 hours after inoculation.
• mice treated with conventional mitoxantrone and liposomal mitoxantrone at 2.5 mg/kg were 13 and 14 days, respectively. This survival time was similar to that described at the same concentration in the single dose study of Example 14. No animals survived to day 60 at this dose level in these treatment groups. Mice treated with liposomal mitoxantrone at 5 mg/kg had a median survival time of 37 days with 4/10 animals surviving to day 60.
• mice bearing xenografted human prostate cancer cells survival was increased after single dose administration of liposomal mitoxantrone, as in Example 7, and mean tumor volume was reduced after multiple dose administration of liposomal mitoxantrone as compared to conventional mitoxantrone-treated animals.
• Male Balb/c, nu/nu, 6-8 week old mice were inoculated with 5 ⁇ 10 6 of human hormone-refractory prostate tumor cells (PC-3). Tumor growth was monitored twice a week until the tumor volumes were in the range of 60-100 mm 2 . Animals were then divided into groups and were treated by i.v.
• This example demonstrates that the liposomal mitoxantrone formulation has a higher concentration in blood plasma, a slower clearance than conventional mitoxantrone following administration to dogs.
• Plasma samples from dogs (3/sex/group) administered conventional mitoxantrone i.v. at 0.13 or 0.26 mg/kg or liposomal mitoxantrone i.v. at 0.26, 0.58 or 0.87 mg/kg were analyzed for mitoxantrone levels by reverse phase HPLC using ametantrone as the internal standard. Time points analyzed were 0, 5 and 30 min and 1, 2, 4, 8 and 24 h after a single dose administration.
• Plasma concentrations in animals receiving conventional mitoxantrone could not be measured at the 5 min time point for the low dose and 30 min for the high dose.
• One male that received 0.258 mg/kg was measurable at the 1 h time point.
• most animals that received liposomal mitoxantrone had mitoxantrone plasma concentrations for up to 2 h for the low dose and 4 h for the mid and high doses.
• This example demonstrates a method for administering liposomal mitoxantrone to patients having cancer and a method for determining a safe and effective amount of a liposomal mitoxantrone formulation.
• Patients with histologically documented solid tumors are selected for treatment.
• the maximum tolerated dose (MTD), dose limiting toxicity, and the blood pharmacokinetics of mitoxantrone following i.v. administration can be determined.
• Anti-tumor effects of liposomal mitoxantrone were also observed.
• Patients are treated with i.v. administration of liposomal mitoxantrone every three weeks until disease progression or occurrence of toxicity requiring early treatment termination was observed.
• the safety and tolerability of treatments are also determined.
• Pharmacokinetic parameters are assessed in the first course of therapy. Cardiac status is evaluated every second course. Disease status is assessed after every second course by appropriate means. Six dose levels are evaluated.
• Adverse events are graded according to NCI/CTC criteria.
• Dose-Limiting Toxicity (DLT) is defined as occurrence within the first course of therapy (i.e. 21 days) of unacceptable toxicity, defined as a grade 3 or 4 nonhematologic toxicity including hypersensitivity reactions, other than nausea/vomiting or alopecia or a grade 4 hematologic toxicity other than neutropenia, or a grade 4 neutropenia which persists for more than 3 days or febrile neutropenia defined as grade 3 or 4 neutropenia with a temperature of greater than 38.5° C., or grade 4 vomiting or grade 4 elevation of hepatic transaminases (AST or ALT), or grade 2 (or higher) decline of LVEF following a MUGA scan.
• DLT Dose-Limiting Toxicity
• the Maximum Tolerated Dose is defined as the highest dose level that causes DLT in no more than one of six patients treated at that level. If none of the initial three patients treated at a given dose level develops dose-limiting toxicity (DLT), dose escalation will continue. If one of the initial three patients treated develops DLT, then three additional patients will be entered on the same dose level. If none of the three additional patients develops DLT, dose escalation will continue. If one or more of the additional three patients treated at a dose level develops DLT, dose escalation will cease. If two or three of the initial three patients treated at a dose level develop DLT, dose escalation will cease. Six patients will be treated at a possible MTD to ensure that criteria are met before declaring that dose level the MTD.
• DLT dose-limiting toxicity
• a subsequent course of treatment may be administered 21 or more days after prior liposomal mitoxantrone dose, and when absolute neutrophil count (ANC) is 1,500 m/m 3 or more and the platelet count is 100,000/mm 3 , and recovery from any other treatment-related toxicity (except alopecia) is to baseline grade or less than grade 1 , whichever is less restrictive.
• ANC absolute neutrophil count
• Treatment is delayed for one week for resolution of toxicities. If toxicities are not resolved after a one-week delay, treatment will be delayed for one additional week, with the same dose reductions as would have occurred after the one-week delay. If treatment must be held for more than two weeks, then the patient will be removed from the study.

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